人破骨细胞抑制因子基因的克隆和表达

Clone and expression of human osteoprotegerin gene

目的 克隆、表达和鉴定人破骨细胞抑制因子(OPG) .方法 从培养的人胚肺成纤维细胞 (WI- 38)中提取总 RNA,经 RT- PCR获得人破骨细胞抑制因子基因 .将该基因克隆到 p GEM3zf-载体中 ,测序鉴定 .继而将 OPG基因插入 TNFHis融合表达载体中 ,4 2℃诱导表达 4～ 5 h,做 SDS-PAGE分析 .以免疫印迹法鉴定 TNFHis OPG融合蛋白的表达 .结果 DNA测序证明 ,获得了 OPG基因 ,其序列与国外文献报道不完全一致 .SDS- PAGE分析表明 ,TNFHis OPG融合蛋白获得表达 ,其 Mr为 7.1× 10 4 ku,表达量约占菌体总蛋白的 10 % .免疫印迹法鉴定显示 ,该融合蛋白可与抗 TNF-α单抗产生阳性反应 .结论 OPG基因的克隆和表达均获得了成功 .

AIM To clone, express and identify human osteoprotegerin (OPG) gene. METHODS Total RNA was extracted from human embryonic lung fibroblast cells (WI 38) and the whole length gene of human OPG was obtained by RT PCR. The OPG gene was cloned into pEGM3zf vector and sequenced. Then the gene was transfered to EcoRI and PstI sites of TNFHis fusion expression vector. After the re combinant bacteria was induced at 42℃ for 4~5 h, the expressed fusion protein (TNFHisOPG) was analyzed by SDS PAGE and western blot. RESULTS DNA sequencing result showed that our OPG gene sequence was not exactly consistent with that reported by some studies. SDS PAGE analysis demonstrated that the TNFHisOPG protein was expressed in E.coli, and the relative molecular mass of TNFHisOPG was 7.1×104 ku. The protein band amounted to 10% of the total bacteria protein and could react with anti TNF α antibody. CONCLUSION Our experimental results showed that OPG gene was successfully cloned and expressed.